Team:UCLA/Notebook/Biobrick

From 2013.igem.org

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<p>To create this biobrick, we utilized a combination of splicing overlap extension PCR and Gibson Assembly to extract a sequence from the BPP-1 phage's genomic DNA that would be free of the standard biobrick restriction enzyme sites. The protocols for those methods are listed below: </p>
<p>To create this biobrick, we utilized a combination of splicing overlap extension PCR and Gibson Assembly to extract a sequence from the BPP-1 phage's genomic DNA that would be free of the standard biobrick restriction enzyme sites. The protocols for those methods are listed below: </p>
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<h4>PCR to generate fragments of <i>mtd</i></h4>
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<h3>PCR to generate fragments of <i>mtd</i></h3>
<p>In this step, four separate fragments of the <i>mtd</i> gene are created via PCR. This step is necessary to eliminate illegal internal biobrick sites (EcoRI, PstI, NotI) and to add necessary biobrick sites to the 5' and 3' ends.</p>
<p>In this step, four separate fragments of the <i>mtd</i> gene are created via PCR. This step is necessary to eliminate illegal internal biobrick sites (EcoRI, PstI, NotI) and to add necessary biobrick sites to the 5' and 3' ends.</p>
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<p>The 20 &#xb5;L PCR mix for each fragment is as follows: </p>
<p>The 20 &#xb5;L PCR mix for each fragment is as follows: </p>
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<p>The thermocycler program for each reaction is as follows: </p>
<p>The thermocycler program for each reaction is as follows: </p>
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<p>The PCR product is then resolved on a 1% agarose gel and excised and column-extracted.</p>
<p>The PCR product is then resolved on a 1% agarose gel and excised and column-extracted.</p>
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<h4>Splicing Overlap-Extension (SOE) PCR to connect fragments 3 and 4</h4>
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<h3>Splicing Overlap-Extension (SOE) PCR to connect fragments 3 and 4</h3>
<p>The 20 &#xb5;L reaction mix is as follows: </p>
<p>The 20 &#xb5;L reaction mix is as follows: </p>
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<h4>Gibson Assembly of Fragments 1,2, and 3-4 </h4>
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<h3>Gibson Assembly of Fragments 1,2, and 3-4 </h3>
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<h4>PCR amplification of <i>mtd</i></h4>
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<h3>PCR amplification of <i>mtd</i></h3>
<p>The mix for the amplification is as follows:</p>
<p>The mix for the amplification is as follows:</p>
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<p>The thermocycler program for the amplification is as follows:</p>
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<tr><th># Cycles</th><th>Temperature (&#176;C)</th><th>Time</th></tr>
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<tr><td>1</td><td>94</td><td>2:00</td></tr>
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<tr><td>35</td><td>98<br>70<br>68</td><td>0:10<br>0:30<br>1:20</td></tr>
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<tr><td>1</td><td>10</td><td>--</td></tr>
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Revision as of 22:53, 27 September 2013




Contents

Making the Mtd Biobrick

To create this biobrick, we utilized a combination of splicing overlap extension PCR and Gibson Assembly to extract a sequence from the BPP-1 phage's genomic DNA that would be free of the standard biobrick restriction enzyme sites. The protocols for those methods are listed below:

PCR to generate fragments of mtd

In this step, four separate fragments of the mtd gene are created via PCR. This step is necessary to eliminate illegal internal biobrick sites (EcoRI, PstI, NotI) and to add necessary biobrick sites to the 5' and 3' ends.

FragmentForward PrimerReverse Primer
1CCTTGAATTCGCGGCCGCATCTAGAATGAGTACCGCAGTCCAGTTCCGGCCTGCGCTGCCGCGTTGCTTCC
2GGAAGCAACGCGGCAGCGCAGGCCCAGCGCCTGGAACTCGTTGTAGTTGGGCAGG
3CCTGCCCAACTACAACGAGTTCCAGGCGCTGGCCGATCCGCGGCCTCCAGTGTTGG
4CCAACACTGGAGGCCGCGGATCGG AAGGCTGCAGCGGCCGCTACTAGTCTCAAGAATCAGG


The 20 µL PCR mix for each fragment is as follows:

ReagentVolume
mtd genomic template1.0 µL (4.5 ng total)
10 µM forward primer1.0 µL
10 µM reverse primer1.0 µL
10 mM dNTPs0.4 µL
Buffer HF4.0 µL
Phusion Polymerase0.2 µL
ddH2O12.4 µL


The thermocycler program for each reaction is as follows:

# CyclesTemperature (°C)Time
1980:30
3098
Variable (70 for fragment 1, 69 for fragment 2, 69 for fragment 3, 72 for fragment 3)
72		0:10
0:20
0:15
1725:00
14--


The PCR product is then resolved on a 1% agarose gel and excised and column-extracted.

Splicing Overlap-Extension (SOE) PCR to connect fragments 3 and 4

The 20 µL reaction mix is as follows:

ReagentVolume or Mass
Fragment 320 ng total
Fragment 420 ng total
10 µM forward primer1.0 µL
10 µM reverse primer1.0 µL
10 mM dNTPs0.4 µL
Buffer HF4.0 µL
Phusion Polymerase0.2 µL
ddH2Ofill to 20 µL

The thermocycler program for this step is as follows:

# CyclesTemperature (°C)Time
1980:30
3098
72
72		0:10
0:20
0:15
1725:00
14--


Gibson Assembly of Fragments 1,2, and 3-4

The mix for the Gibson Assembly is as follows:

ReagentConcentration or Volume
Fragment 10.2 pM
Fragment 20.2 pM
Fragment 3-40.2 pM
Assembly Master Mix (from New England Biolabs)fill to 5.0 µL

The mix is then incubated at 50 °C for 1 hour.


PCR amplification of mtd

The mix for the amplification is as follows:

ReagentVolume or Mass
mtd20 ng
10 µM forward primer (CCTTGAATTCGCGGCCGCATCTAGAATGAGTACCGCAGTCCAGTTCCG )0.6 µL
10 µM reverse primer (AAGGCTGCAGCGGCCGCTACTAGTCTCAAGAATCAGG )0.6 µL
10 mM dNTPs0.4 µL
2x xtreme buffer10 µL
KOD xtreme hot start polymerase0.4 µL
ddH2Ofill to 20 µL

The thermocycler program for the amplification is as follows:

# CyclesTemperature (°C)Time
1942:00
3598
70
68		0:10
0:30
1:20
110--

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